Cell size regulates human endoderm specification through actomyosin-dependent AMOT-YAP signaling

Summary Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.

(G) Cell size distribution of AMOT knockdown and control cells, determined by FSC.

Gene Forward primer
Reverse primer

Immunofluorescence assay and alkaline phosphatase (AP) staining
For immunofluorescence assay, cells were fixed in 4% paraformaldehyde after PBS washing and blocked with blocking solution (DPBS with 10% (v/v) donkey serum and 0.3% Triton-100).Then cells were incubated overnight with the primary antibodies at proper concentration at 4 °C.The used primary antibodies in the experiment were:

RNA extraction and RT-qPCR
Total RNA from cells was extracted with HiPure Total RNA Mini Kit (Magen, Cat#R4111-03) according to manufacturer's manual. 1 mg of total RNA was used for reverse-transcription to cDNA with the ABScript II RT Master Mix (ABclonal, Cat#RK20402).RT-qPCR was performed on a C1000 Touch Thermal Cycler machine (Bio-Rad) using 2x SYBR Green qPCR Master Mix (Biomake, Cat#B21203).The relative gene expression levels were normalized to the level of GAPDH based on the delta Ct method.All RT-qPCR experiments were carried out at least three replicates.
Comparison between samples was performed using Student's t test.The primers used in the RT-qPCR assays are listed in Table S1.

RNA-seq and data analysis
RNA was isolated with HiPure Total RNA Mini Kit and sent to Geekgene (Beijing, China) for RNA-seq library construction and sequencing on an Illumina Hiseq X Ten platform with paired-end reads.For data analysis, RNA-seq raw data that contained adapters were removed and trimmed by Trim Galore (v0.(https://biit.cs.ut.ee/gprofiler/gost),Venn diagram was made by Venn website (https://bioinformatics.psb.ugent.be/webtools/Venn/)and the heatmap was made by pheatmap (v1.0.12).

AMOT knockdown
We utilized shRNA for knockdown and the shRNAs specifically against AMOT and scramble control were cloned into lentiviral vector pLKO.1 plasmid (Addgene Plasmid 10878).For lentivirus packaging, the lentiviral shRNA plasmid and lentiviral helping vectors (psPAX2, pMD2.G) were transfected in HEK293T cells and the virus was concentrated.After lentivirus infection to human ESCs, 2 mg/mL puromycin was used for selection to establish stable shAMOT knockdown ESC line.The sequences of oligoes were listed as Fig. S7A and below: shControl: GAAGTATTCCGCGTACGTT.

Statistical analysis
Statistical analysis was conducted using PRISM 9.5.1 for macOS.The results were shown as means ± SD from at least three independent experiments.Single comparison between two groups was analyzed by two-tailed unpaired t-test.Comparisons between multiple groups were determined using One-Way ANOVA analysis.Pearson's correlation analysis was used to evaluate the correlation between two variables.P value<0.05 is considered statistically significant (* means p < 0.05, ** means p < 0.01, and *** means p < 0.001), while "n.s." stands for not statistically significant.

Figure S1 .
Figure S1.Differentiation induces changes of cell volume and mechanical state.

Figure S2 .
Figure S2.Hypertonic pressure induces changes of cell volume and enhances human endoderm differentiation.

Figure S4 .
Figure S4.Cytoskeletal plays a role in endoderm differentiation boost caused by cell size decrease.

Figure S5 .
Figure S5.Identification of cell size related signal pathway that contributes to hypertonic endoderm differentiation.

Figure
Figure S7.AMOT is not essential for pluripotency but influential for YAP activation.

Figure S1 .
Figure S1.Differentiation induces changes of cell volume and mechanical state.

Figure S2 .
Figure S2.Hypertonic pressure induces changes of cell volume and enhances human endoderm differentiation.

Figure S5 .
Figure S5.Identification of cell size related signal pathway that contributes to hypertonic endoderm differentiation.

Figure
Figure S7.AMOT is not essential for pluripotency but influential for YAP activation.
Cells were fixed according to the manufacturer's instructions of Transcription Factor Buffer Set (BD, #562574) and then incubated with SOX17-Alexa488 (BD, #562205) antibody.Corresponding isotype was used as control.The SOX17-positive or CXCR4positive cells were detected by NovoCyte flow cytometer (ACEA, USA) and analyzed by FlowJo software.For cell cycle analysis, a cell cycle detection kit (Keygen, China, Cat#KGA512) was used according to the protocol included in the kit.DE cells were dissociated into single cells and fixed with chilled anhydrous ethanol for 1 hour at room temperature.After washed with phosphate-buffered saline (PBS) twice, cells were resuspended with 500μL RNase A solution (100μg/ml RNase A) and incubated at 37 °C for 30 minutes.Then the cells were added with propidium iodide (PI) solution (50μg/ml PI) and incubated at room temperature for another 30 minutes in dark.Finally, a NovoCyte flow cytometer (ACEA, USA) was used to analyze these cells.The DNA content was analyzed on the basis of PI intensity in the FlowJo (v10.4.0), by which these sorted cells were divided into each cell cycle phase (subG1, G1, S, and G2/M) and calculated the proportion of each phase.For apoptosis analysis, an Annexin V-FITC/PI apoptosis detection kit (Keygen, China, Cat#KGA106) was used.DE cells were dissociated into single cells and added with Annexin V-FITC/PI, followed with incubation at room temperature for 15 minutes in dark.Cell apoptosis should be assessed by flow cytometer within 1 hour and later analyzed by FlowJo.